Single cycle replicating adenovirus vectors

ABSTRACT

This document relates to adenovirus vectors and methods and materials related to using adenovirus vectors. For example, viruses, nucleic acid molecules encoding viruses, cell lines containing viral vectors, and methods for using viruses to deliver nucleic acid to cells in vitro or in vivo are provided. Methods and materials for using adenovirus vectors to induce immune responses and to treat cancer also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/863,705, filed Apr. 30, 2020 (now U.S. Pat. No. 11,279,953), which isa continuation of U.S. application Ser. No. 16/570,535, filed Sep. 13,2019 (now U.S. Pat. No. 10,640,786), which is a continuation of U.S.application Ser. No. 16/153,880, filed Oct. 8, 2018 (now U.S. Pat. No.10,465,206), which is a divisional of U.S. application Ser. No.12/920,775, filed Sep. 2, 2010 (now U.S. Pat. No. 10,131,921), which isa National Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2009/036392, filed Mar. 6, 2009, which claimspriority from U.S. Provisional Application Ser. No. 61/068,287, filedMar. 6, 2008. The disclosures of the prior applications are consideredpart of (and are incorporated by referenced in) the disclosure of thisapplication.

TECHNICAL FIELD

This document relates to adenovirus vectors and methods and materialsrelated to using adenovirus vectors.

BACKGROUND INFORMATION

Adenoviruses can be easy to grow and manipulate, and they can exhibitbroad host range in vitro and in vivo. The adenovirus life cycle doesnot require integration into the host cell genome, and foreign genes canbe delivered using adenovirus vectors. For example, adenovirus vectorswere successfully used in eukaryotic gene expression and vaccinedevelopment (Casimiro et al., J. Virol., 77:6305-6313 (2003); Benson etal., J. Virol., 72:4170-4182 (1998); Buge et al., J. Virol.,73:7430-7440 (1999); and Robert-Guroff et al., J. Virol., 72:10275-10280(1998)).

SUMMARY

This document relates to adenovirus vectors and methods and materialsrelated to using adenovirus vectors. For example, this document providesviruses, nucleic acid molecules encoding viruses, cell lines containingviral vectors, and methods for using viruses to deliver nucleic acid tocells in vitro or in vivo. This document also provides methods for usingadenovirus vectors to induce immune responses and to treat cancerpatients. In some cases, single cycle replicating adenoviruses can beused to deliver antigens that trigger an immune response within amammal.

The nucleic acid molecules and viruses provided herein can lack all or aportion of the sequence encoding an adenovirus fiber protein and/or anadenovirus V protein and can contain a nucleic acid sequence thatencodes one or more polypeptides that are heterologous to naturallyoccurring adenoviruses.

This document is based, in part, on the discovery that adenoviruses canbe modified to create single cycle replicating adenoviruses. Thisdocument also is based, in part, on the discovery that single cyclereplicating adenoviruses can be used to deliver nucleic acid encodingantigens such that a mammal produces an effective immune responseagainst those antigens.

In general, one aspect of this document features a method for inducingan immune response against an antigen in a mammal. The method comprises,or consists essentially of, administering an adenovirus to the mammalunder conditions wherein the adenovirus infects a cell of the mammal,wherein the adenovirus comprises an adenovirus polypeptide and lacks atleast a portion of a nucleic acid sequence that encodes the adenoviruspolypeptide, wherein the adenovirus comprises a nucleic acid sequenceencoding the antigen, and wherein expression of the antigen in the cellleads to induction of the immune response. The mammal can be a human.The antigen can be a tumor antigen. The tumor antigen can be NY-ESO,EBV-LMP, or a papilloma virus antigen. The cell can be an epithelialcell, a tumor cell, a hematopoietic cell, or an antigen presenting cell.The adenovirus polypeptide can be a fiber protein, a V protein, hexon,penton-base, or pIII.

In another aspect, this document features a method for delivering atherapeutic polypeptide to a mammal. The method comprises, or consistsessentially of, administering an adenovirus to the mammal underconditions wherein the adenovirus infects a cell of the mammal, whereinthe adenovirus comprises an adenovirus polypeptide and lacks at least aportion of a nucleic acid sequence that encodes the adenoviruspolypeptide, wherein the adenovirus comprises a nucleic acid sequenceencoding the therapeutic polypeptide. The mammal can be a human. Thetherapeutic polypeptide can be a thymidine kinase, a fusogenicglycoprotein, a tumor suppressor p53, a heat shock protein polypeptide.The cell can be a tumor cell, a hematopoietic cell, a liver cell, or agastrointestinal cell. The adenovirus polypeptide can be a fiberprotein, a V protein, hexon, penton-base, or pIII.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic diagram of the Ad-rep-GFP-Luc vector. FIG. 1B isa graph plotting GFP expression after infection with the indicated virusparticle/cell ratios on a microplate reader.

FIG. 2A is a graph plotting muscle luciferase activity in mice infectedwith replication-defective or replication-competent Ad5 vectors on theindicated day post infection. 1×10¹⁰ virus particles of the indicatedGFP-Luc expressing viruses were administered intramuscularly into mice.The mice were imaged on a Lumazone for luciferase activity on theindicated days. The results are shown for the mean photons for theinjection site indicating expression of the luciferase transgene in thetissues. * indicates p value of 0.0276 for last time point. FIG. 2B is abar graph plotting anti-luciferase antibody responses for BALB/c miceimmunized intramuscularly (i.m.) or intranasally (i.n.) with theindicated vectors. The antibody responses against luciferase weremeasured from serum 3 weeks later. Significance was 0.0026 by one wayANOVA. * and ** indicate p values of 0.038 and 0.0008, respectively, byBonferroni's multiple comparison test.

FIG. 3A contains a schematic diagram of a Ad-Blue-ΔF vector. Expressionof the Ad-Blue-ΔF vector was detected by fluorescent imaging of BFP intransfected cells. FIG. 3B contains a schematic diagram of a Ad-Red-ΔVvector and photographs of cells transfected with the viral genomes at 2and 21 days. dsRed expression was detected in cells transfected witheach vector by the detection of red fluorescence. Brightfield imagesdemonstrated that the deleted-fiber vector grows efficiently when fiberis supplied from the cell line. Unlike wild-type virus with intactprotein V, the ΔV vector does not form red plaques after 3 weeksindicating that the deletion has rendered the virus defective for virusproduction and spread.

FIG. 4 contains Western blot results of wild-type and codon-optimizedfiber expression in the indicated cells for use as helper cells toproduce fiber-deleted viruses.

FIG. 5 contains Western blot results of cesium chloride (CsCl) purifiedwild-type Ad5 vector (A) and Ad5 with fiber deletion (C) expressed intransfected 293 cells. An irreverent Ad5 modified with short and longfibers from Ad41 is also shown (B). Western blotting was performed usingan antibody that recognizes the N-termini of the fiber proteins.

FIG. 6 is a sequence listing for a nucleic acid sequence (SEQ ID NO:1)encoding a codon-optimized fiber protein.

DETAILED DESCRIPTION

This document provides viruses (e.g., single cycle replicatingadenoviruses), nucleic acid molecules encoding viruses, cell linescontaining viral vectors, methods for using viruses to deliver nucleicacid to cells in vitro or in vivo, methods for using adenovirus vectorsto induce immune responses, and methods for using adenovirus vectors totreat cancer patients.

This document provides nucleic acid molecules that can encode singlecycle replicating adenoviruses. Nucleic acid molecules encoding singlecycle replicating adenoviruses can include all the naturally-occurringsequences of an adenovirus (e.g., an Ad5 virus) with the exception thatit lacks all or a portion of at least of one of the following adenovirussequences: fiber protein-encoding sequence, V protein-encoding sequence,hexon-encoding sequence, penton base-encoding sequence, VA RNA-encodingsequence, pill protein-encoding sequence, or other early or late geneproduct-encoding sequences. Examples of adenoviral nucleic acidsequences that encode polypeptides include, without limitation, thoseset forth in GenBank gi numbers 209842, 58478, or 2935210, and/orannotated in GenBank accession numbers M73260, X17016, or AF030154.

In some cases, a deletion of all or a portion of the nucleic acidencoding one or more of the following polypeptides can be engineeredinto a nucleic acid encoding an adenovirus: fiber protein-encodingsequence, V protein-encoding sequence, hexon-encoding sequence, pentonbase-encoding sequence, VA RNA-encoding sequence, pIII protein-encodingsequence, or other early or late gene product-encoding sequences. Suchdeletions can be any length that results in the deletion of one or moreencoded amino acids. For example, portions of a nucleic acid sequence ofan adenovirus can be removed such that an encoded polypeptide lacks 5,6, 7, 8, 9, 10, 15, 20, 25, 30, or more amino acid residues). Theportion or portions to be deleted can be removed from any location alongthe length of the sequence. For example, a portion of an adenovirusnucleic acid sequence can be removed at the 5′ end, the 3′ end, or aninternal region of an adenovirus nucleic acid such as a fiberprotein-encoding sequence, V protein-encoding sequence, hexon-encodingsequence, penton base-encoding sequence, VA RNA-encoding sequence, pIIIprotein-encoding sequence, or other early or late gene product-encodingsequences.

Any appropriate molecular biology and biochemical method (e.g., nucleicacid sequencing) can be used to identify the presence and location of adeletion introduced into an adenovirus nucleic acid. For example,nucleic acids can be separated by size using gel electrophoresis toconfirm that portions have been removed relative to the length of theoriginal nucleic acid. In some cases, antibodies that recognize variousepitopes on the encoded polypeptide (e.g., a fiber polypeptide) can beused to detect the presence or absence polypeptides targeted fordeletion.

In some cases, a nucleic acid molecule provided herein can include asequence encoding a therapeutic polypeptide or an immunogen. Examples oftherapeutic polypeptides include, without limitation, propionyl CoAcarboxylase (NM 000282; gi number 5095), dystrophin (M92650; gi number1756), p53 (M14695), factor IX (BC109215; gi number 7157), herpes virusthymidine kinase (NC 001798), measles H and F fusogenic glycoproteins(DQ227321; gi number 1489803), sodium iodide symporter (NM_000453; ginumber 6528), and heat shock protein (L12723; gi number 3308).Expression of such therapeutic polypeptides can be driven by, forexample, linking polypeptide encoding sequences to constitutive,inducible, and/or tissue-specific promoter sequences that can drivetranscription of a therapeutic polypeptide. Examples of immunogensinclude, without limitation, cancer immunogens such as melanoma antigenfamily A (NM_004988; gi number 4100), MUC1 (Z17325; gi number 4582),Epstein-Barr virus LMP-2 (V01555), and human papilloma virus proteins(K02718), viral immunogens such as HIV gag and env (NC 001802),influenza hemagglutinin (EU497921), hepatitis C core (NC 004102; ginumber 951475), and smallpox H3L antigen (DQ437592), and bacterialimmunogens such as Bacillis anthracis protective antigen (AF268967) andmecA from Staphylococcus aureus (AB353125). In some cases, an immunogencan be a full-length immunogenic polypeptide or a portion thereof. Forexample, a nucleic acid sequence encoding an immunogenic polypeptide canbe modified to remove portions of nucleic acid such that the encodedpolypeptide lacks any number of amino acids (e.g., 5, 10, 15, 20, 30amino acids, or all amino acids of the immunogenic polypeptide). In somecases, portions of a nucleic acid sequence encoding an immunogenicpolypeptide can be removed from anywhere along the length of thesequence. For example, portions of the nucleic acid sequence can beremoved at the 5′ end, the 3′ end, or an internal region of the targetnucleic acid. In some cases, a therapeutic polypeptide and/or animmunogenic polypeptide can be designed to be secreted from cellsinfected with the adenovirus encoding the therapeutic polypeptide and/orimmunogenic polypeptide.

Any appropriate method can be used to detect expression of a therapeuticpolypeptide or immunogenic polypeptide from virus infected cells. Forexample, antibodies that recognize a therapeutic polypeptide orimmunogenic polypeptide can be used to detect the presence or absence ofthe polypeptide in infected cells.

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. A nucleic acid can be double-stranded orsingle-stranded. A single-stranded nucleic acid can be the sense strandor the antisense strand. In addition, a nucleic acid can be circular orlinear.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that are present in a viral genome,including nucleic acids that normally flank one or both sides of thenucleic acid in a viral genome. The term “isolated” as used herein withrespect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., any paramyxovirus,retrovirus, lentivirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not considered an isolated nucleicacid.

As described herein, a nucleic acid molecule can encode an adenoviruswith the exception that it lacks all or a portion of at least of oneadenovirus polypeptide-encoding sequence. Any appropriate molecularcloning technique (e.g., recombination or site-directed mutagenesis) canbe used to generate an adenovirus nucleic acid molecule that lacks allor a portion of a fiber protein-encoding sequence, a V protein-encodingsequence, hexon protein-encoding sequence, penton base protein-encodingsequence, VA RNA-encoding sequence, or pIII protein-encoding sequence.Likewise, any appropriate molecular cloning technique (e.g., PCR,recombination, or restriction site cloning) can be used to introduce anucleic acid sequence into a nucleic acid molecule of an adenovirus. Thenucleic acid molecules provided herein can be incorporated into virusesby standard techniques. For example, recombinant techniques can be usedto insert a nucleic acid molecule provided herein into an infectiveviral genome or sub-genome within a plasmid or other vector. In somecases, a plasmid or other vector can additionally express luciferase oranother reporter gene. The viral genome can then be transfected intomammalian cells to rescue the modified adenovirus. Alternately, modifiedsubgenome sequences can be co-transfected into cells with othersubgenome sequence such that the mammalian cells recombines thesubgenomes into an intact genome making new virus.

The document also provides viruses (e.g., adenoviruses) containing anucleic acid molecule provided herein. For example, this documentprovides recombinant adenoviruses that lack all or a portion of a fiberprotein-encoding sequence, a V protein-encoding sequence, a hexonprotein-encoding sequence, a penton base protein-encoding sequence, a VARNA-encoding sequence, a pIII protein-encoding sequence, or other earlyor late gene product-encoding sequence. In some cases, such adenovirusescan contain the polypeptide while lacking all or a portion of thenucleic acid sequence that encodes that polypeptide. For example, anadenovirus provided herein can be contain adenovirus fiber protein whilelacking the nucleic acid that encodes adenovirus fiber protein. Suchadenoviruses can be obtained using cells lines designed to express thepolypeptide such that it is incorporated into virus particles that lackthe nucleic acid sequence that encodes that polypeptide.

This document provides cell lines that can be used to produce viruses(e.g., adenoviruses) that contain a particular polypeptide while lackingthe nucleic acid sequence that encodes that polypeptide. For example,this document provides isolated cells that express an adenovirus fiberprotein, an adenovirus V protein, an adenovirus hexon protein, anadenovirus penton base protein, an adenovirus VA RNA, an adenovirus pIIIprotein, or other adenovirus early or late gene product. Such cells cancontain nucleic acid that allows adenovirus particles to be producedsuch that they contain the expressed adenovirus polypeptide, whilelacking the nucleic acid sequence that encodes that polypeptide.

The viruses provided herein can be used to induce immune responseswithin a mammal and/or can be used to treat cancer. For example, anadenovirus can be designed to be a single cycle replicating adenovirus(e.g., a fiber protein-less or V protein-less adenovirus) that containsnucleic acid that drives expression of a cancer immunogen. Such anadenovirus can be propagated in a cell that provides the missingadenovirus polypeptide (e.g., fiber protein-expressing cell line or Vprotein-expressing cell line) in order to increase the available numberof copies of that virus, typically by at least 100-fold (e.g., by100-fold to 15,000-fold, by 500- to 10,000-fold, by 5,000- to10,000-fold, or by 5,000- to 15,000-fold). A virus can be expanded untila desired concentration is obtained in standard cell culture media(e.g., DMEM or RPMI-1640 supplemented with 5-10% fetal bovine serum at37° C. in 5% CO₂). A viral titer typically is assayed by inoculatingcells (e.g., A549 or 293 cells) in culture or by quantitating viralgenomes by optical density or real-time PCR.

Viral stocks can be produced by growth in mammalian cells. Viral stockscan be aliquoted and frozen, and can be stored at −70° C. to −80° C. atconcentrations higher than the therapeutically effective dose. A viralstock can be stored in a stabilizing solution. Examples of stabilizingsolutions include, without limitation, sugars (e.g., trehalose,dextrose, glucose), amino acids, glycerol, gelatin, monosodiumglutamate, Ca²⁺, and Mg²⁺.

The viruses provided herein can be administered to a mammal to deliveran encoded therapeutic polypeptide or to induce an immune responseagainst an encoded immunogen. For example, a composition containing avirus provided herein can be administered to a mammal by, for example,subcutaneous, intramuscular, intravenous, intratumoral, or oraladministration. In some cases, the viruses provided herein can bedesigned to induce an anti-cancer immune response. Examples of types ofcancers that can be treated using the viruses provided herein include,without limitation, myeloma, melanoma, glioma, lymphoma, leukemia, andcancers of the lung, brain, stomach, colon, rectum, kidney, prostate,ovary, and breast. For example, an adenovirus lacking fiberprotein-encoding nucleic acid and containing a gp100 or fusogenicglycoprotein polypeptide fragment can be used to treat melanoma.

The viruses provided herein can be administered to a mammal (e.g., ahuman) in a biologically compatible solution or a pharmaceuticallyacceptable delivery vehicle. Suitable pharmaceutical formulations dependin part upon the use and the route of entry, e.g., transdermal or byinjection. Such forms should not prevent the composition or formulationfrom reaching target cells (e.g., immune cells, or tumor cells) or fromexerting its effect. For example, pharmacological compositions injectedinto the blood stream should be soluble.

While dosages administered can vary from patient to patient (e.g.,depending upon desired response or the disease state), an effective dosecan be determined by setting as a lower limit the concentration of virusproven to be safe and escalating to higher doses, while monitoring forthe desired response (e.g., antibody production, T cell responses, areduction in cancer cell growth, or reduction in cancer-associatedprotein) along with the presence of any deleterious side effects.

The viruses provided herein can be delivered in a dose ranging from, forexample, about 10³ pfu to about 10¹² pfu (typically >10⁸ pfu). Atherapeutically effective dose can be provided in repeated doses. Repeatdosing is appropriate in cases in which observations of clinicalsymptoms or tumor size or monitoring assays indicate either that a groupof cancer cells or tumor has stopped shrinking or that the degree ofviral activity is declining while the tumor is still present. Repeatdoses (using the same or a different modified virus) can be administeredby the same route as initially used or by another route. Atherapeutically effective dose can be delivered in several discretedoses (e.g., days or weeks apart) and in some cases, one to about twelvedoses are provided. In some cases, a therapeutically effective dose ofviruses can be delivered by a sustained release formulation.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Comparing Replication-Competent Vs.Replication-Defective Adenoviruses

To determine whether replicating adenovirus vectors generate betterimmune responses than replication-defective adenovirus vectors afteroral delivery, replication-competent Ad5 was modified to over-expresstransgenes by inserting a CMV-GFP-luciferase expression cassette at aHpaI site between E1a and E1b (FIG. 1A). In vitro comparison of GFPfluorescence between replication-competent (Ad-rep-GFP-Luc) andreplication-defective Ad in vitro demonstrated that thereplication-competent vector expressed transgenes to a much higher levelat equal virus particles/cell (FIG. 1B).

When the Ad-rep-GFP-Luc replication-competent vector was tested in vivoin nude mice bearing human tumors, luciferase imaging revealed the virusfirst seeds the liver, replicates, and then expands into tumors (FIG.1C). Direct comparison of luciferase activity after intramuscularinjection demonstrated that the replication-competent vector producedless luciferase initially, but that transgene expression appeared toincrease over time in contrast to the replication-defective virus (FIG.2A). This increase was not observed after intranasal administration.Imaging captures only the strongest localized signals, however, anddispersed lower expression may not be captured. When immune responseswere compared, it was demonstrated that the replication-competentvectors generated substantially higher immune responses against thetransgene products by both routes (FIG. 2B), indicating that the virusesreplicate sufficiently in the mouse model to evaluate their utility asvaccine carriers.

These results demonstrate that replicating adenovirus vectors such asAd-rep-GFP-Luc can be used as delivery vectors. These results alsodemonstrate that replicating adenovirus vectors can be used to induceeffective immune responses in mammals.

Example 2—Single Cycle Replicating Adenoviruses Based on Deletion of theLate Gene for the Fiber Protein

The adenovirus fiber protein mediates binding to its receptor CAR(Campos and Barry, Curr. Gene Ther., 7:189-204 (2007)). Viruses lackingfiber are defective due to loss of CAR binding and due to incompletematuration of virus particles. The viral genome plasmid Ad-Blue-ΔF wasconstructed in which the entire fiber coding sequence was replaced witha zeocin-resistance marker by recombination in bacteria (FIG. 3A). Thisvirus carried the blue fluorescent protein (BFP) gene in the E3 regionthat is expressed only in replicating virions to mark successfulreplication of the vector. When this fiber-deleted genome wastransfected into 293 cells, western blot analysis demonstrated that thisvector expresses no fiber proteins. When this virus was propagated in293 cells, it failed to spread to other cells (FIG. 3A). In contrast,when the same genome was transfected into fiber-expressing 633 cells,virus production was rescued. Therefore, a single-cycle Ad vector can beproduced in fiber-expressing 633 cells, but they cannot spread afterinitial infection because they lack the fiber gene. By growingAd-Blue-ΔF in 633 cells, normal yields of virions displaying fibers wereproduced, but they did not contain nucleic acid encoding fibers. Bygrowing Ad-Blue-ΔF in 633 cells and then 293 cells in the last round ofamplification, Ad-Blue-ΔF without fiber were produced in normal yields(e.g. 5×10¹² virus particle preps). Therefore, this system can be usedto make large batches of Ad vaccines displaying fiber proteins, but thatcannot produce new virions with fibers after infection in vivo.

Example 3—Single Cycle Replicating Adenoviruses Based on Deletion of theEarly Gene for Protein V

Protein V is a 42 kDa protein present at 160 copies per Ad virion(Campos and Barry, Curr. Gene Ther., 7:189-204 (2007)). The followingwas performed to test whether protein V can be deleted from Ad5 byknocking the gene out in a dsRed-expressing vector by homologousrecombination in bacteria (FIG. 3B). When tested in 293 cells, Ad-Red-AVproduced reporter gene product, but never plagued (FIG. 3B). Theseresults demonstrate that viruses containing a deleted protein V can beused to generate single-cycle replicating viruses. In this case,deletion of protein V targets an early gene product rather than the latefiber gene. AV viruses produced in cell lines expressing protein V intrans (like fiber-expressing 633 cells) can encapsidate protein V,allowing progeny virions to infect and transduce cells in vivo.Following infection as a vaccine in vivo, viral ΔV genomes can bereplicated normally. However, progeny virions would lack protein V andwould be unable to infect actively a second set of cells after theinitial infection.

In summary, these results demonstrate that, unlike areplication-competent Ad, single-cycle fiberless or protein V deleted Adprogeny virions can be infectious only when administered. Secondaryvirions cannot propagate an uncontrolled adenovirus infection like liveAd4, 5, and 7 vaccines and cannot be spread or cause disease in peoplein close or distant contact with the vaccine or person receiving thetherapeutic. Unlike replication-defective Ad vaccines, fiber-deleted Adproduced from 633 cells or protein V-deleted virions can replicate theirgenomes and antigen genes 10,000-fold in infected cells. Thesesingle-cycle viruses therefore can be used to amplify antigen productionwithout the risk of causing uncontrolled adenovirus infections.

Example 4—Codon-Optimized Fiber Expression

Western blot analysis was performed to test the use of codon-optimizedfiber expression in various cell lines for use as helper cells toproduce fiber-deleted viruses. The nucleic acid sequence encoding acodon-optimized fiber protein used for the expression assays was as setforth in SEQ ID NO:1. As demonstrated in FIG. 4 , transiently- andstably-transfected cells can produce fiber-deleted viruses. Fluorescentimaging of transfected 293 cells revealed robust plaque formation ofBFP-expressing Ad with fiber-deletion in 293 cells stably expressingcodon-optimized fiber, but poor plaque formation in 293 cells lackingfiber. These results demonstrate that cells expressing codon-optimizedfiber can be used to propagate viruses with fiber-deletion genomes.These results also demonstrate that cells expressing codon-optimizedfiber can be used to establish fiber libraries.

To assess fiber protein incorporation into Ad viruses with afiber-deletion genome, western blot analysis was performed on virusesproduced by infection of 293 cells with Ad5 vector (FIG. 5A), byinfection of codon-optimized fiber-expressing 293 cells with Ad5 withfiber-deletion (FIG. 5C), or by infection of 293 cells with anirreverent Ad5 modified with short and long fibers from Ad41 (FIG. 5B).Viruses were purified by banding in a cesium chloride (CsCl) gradient.Western blots were performed using an antibody that recognized theN-terminus of the fiber protein. Western blot data revealed that whenthe fiber-deleted Ad5 virus was propagated in codon-optimizedfiber-expressing 293 cells, the cells produced sufficient levels offiber protein for normal fiber incorporation into Ad virions (FIG. 5 ).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for inducing an immune response againstan antigen in a mammal, wherein said method comprises: (a) providing aninitial administration of a single cycle replicating adenovirus to saidmammal under conditions wherein said adenovirus infects a cell of saidmammal and replicates within said cell, wherein said cell does notproduce new infectious virions following infection with said adenovirus,wherein said adenovirus comprises an adenovirus polypeptide and lacks atleast a portion of a nucleic acid sequence that encodes said adenoviruspolypeptide, wherein when said adenovirus lacks at least a portion of anucleic acid sequence that encodes a fiber protein, said adenoviruscomprises a fiber protein having wild-type specificity, wherein saidadenovirus comprises a nucleic acid sequence encoding said antigen, andwherein expression of said antigen in said cell leads to induction ofsaid immune response, and (b) providing a repeated administration ofsaid single cycle replicating adenovirus to said mammal.
 2. The methodof claim 1, wherein said mammal is a human.
 3. The method of claim 1,wherein said antigen is a tumor antigen.
 4. The method of claim 3,wherein said tumor antigen is NY-ESO, EBV-LMP, or a papilloma virusantigen.
 5. The method of claim 1, wherein said cell is an epithelialcell, a tumor cell, a hematopoietic cell, or an antigen presenting cell.6. The method of claim 1, wherein said adenovirus polypeptide is a fiberprotein, a V protein, hexon, penton-base, or pIII.